Summary: Nitric oxide effect on platelets from sickle cell patients: Sickle cell anemia is a disease associated with periodic attacks of ishemia and thrombosis. The underlying etiology is a defect in the hemoglobin that leads to red cell sickling in conditions of hypoxia. Nitric oxide is thought to be involved in the pathophysiology of this disease and could potentially be used as therapy. We found that platelets from patients with sickle cell anemia were more sensitive to pharmacologic nitric oxide donors when compared to normal platelets. These observations will be useful in selecting particular nitric oxide donors and in understanding the effect of nitric oxide therapy in sickle cell patients. Mechanisms of adenovirus-induced thrombocytopenia: The use of adenovirus, as a vector for gene therapy in a recent clinical trial in Ornithine Transcarbamylase deficient patients, was associated with thrombocytopenia. In most cases the drop in platelets was moderate and reversible with a single exception where thrombocytopenia was also associated with a significant coagulopathy and death. The etiology of the adenovirus-associated thrombocytopenia is not clear but may be a serious complication of gene therapy. Viral entry into cells involves two independent steps. The first step is attachment of virus to a receptor, in the case of adenovirus, this receptor is coxsackievirus adenovirus receptor, also known as CAR. The second step of infection involves entry into the host cell and is mediated by a co-receptor. For adenovirus, this co-receptor is an av integrin. Integrins are heterodimeric protein complexes found on many cell types; they bind adhesive proteins containing the recognition sequence arginine-glycine-aspartic acid (RDG). Examples of RGD-containing proteins include fibrinogen, fibronectin, vitronectin, and laminins. A number of integrin molecules are expressed on platelets and these include glycoprotein IIb/IIIa, the fibrinogen receptor and the vitronectin receptor. Conformational changes in the glycoprotein Gp IIb/IIIa , induced by activation of platelets by agonists or by binding of monoclonal antibodies to this glycoprotein, can lead to fibrinogen binding to platelets and to platelet aggregation. Since adenovirus interacts with integrins it could potentially induce or interfere with fibrinogen binding to platelets and produce platelet aggregates in vivo which would be cleared from circulation. We have investigated whether adenovirus has a direct effect on platelets in vitro by observing the effect of adenovirus on agonist induced platelet aggregation. The presence of adenovirus did not inhibit or potentiate platelet in vitro responses to agonists. Since a direct effect of the virus on platelets did not appear to explain the mechanism of thrombocytopenia evidenced in clinical trials, we investigated this mechanism in vivo. In a collaborative study, we followed platelet counts and circulation survival in rhesus monkeys given high doses of clinical grade, replication incompetent, adenovirus intravenously. Platelet survival in circulation was determined with ex vivo biotin labeling of platelets. reinfusion of the labelled platelets prior to administration of adenovirus and then determination of the number of labelled platelets remaining in circulation by flow cytometry. Injection of adenovirus caused a dose dependent clearance of platelets from circulation with minimal effect on platelet production. Further work will be conducted to elucidate the molecular mechanism of adenovirus induced thrombocytopenia. Effects of cold temperature storage on platelets: The risk of bacterial contamination could be reduced if platelets were stored in cold temperatures but cold exposure has a negative impact on the survival of platelets in circulation after transfusion. Moreover, such conditions potentiate the responsiveness of platelets to certain agonists in platelet aggregation although it is not clear whether this contributes to the shortened circulation times. We are investigating the mechanisms involved in the potentiation of aggregation responses. Cold temperatures exposure does not increase agonist-induced fibrinogen binding or increase the number of fibrinogen binding sites. However, aggregates of cold exposed platelets are more resistantant to disaggregating agents suggesting that this resistance may be the mechanism for the potentiated aggregation response.